The present invention relates to oligonucleotide synthesis, and more particularly to in situ synthesis of oligonucleotides of inverse orientation.
Oligonucleotide Arrays
Insights in the genetic make-up of man and other organisms increases rapidly. Moreover, information about the role of specific genes in diseases also accumulates at a rapid rate. Accordingly, there is a growing need for methods to analyse large sets of genetic factors in parallel. Oligonucleotide arrays, that is sets of oligonucleotides distributed in a two-dimensional pattern on the surface of a planar device, are promising as a means to study many nucleotide positions in a target DNA or RNA molecule. They can also be used to determine relative copy numbers or the presence of sequence variants of several different nucleic acid sequences in a sample. Numerous research groups have contributed to the development of methods for efficient construction of arrays, means to record the outcome of sample analyses, and for computation of the results.
Two principally distinct approaches have been taken for the construction of such oligonucleotide arrays. Individual oligonucleotides may be manufactured separately, purified, and characterized before they are immobilized in defined patches on a planar solid phase. Techniques used for this purpose include deposition via ink jet printing or direct transfer of liquid oligonucleotide samples with pen-like devices. These methods allow good control of the quality of the reagents immobilized, but arrays of high complexity, that is with more than around 1000 different specificities, are difficult to manufacture.
The other major approach to construct arrays is through in situ synthesis, where the stepwise synthesis of oligonucleotides is performed directly on the devices (1), (2). Typically, oligonucleotide synthesis proceeds from the 3xe2x80x2-end towards the 5xe2x80x2-end of the probe molecule. As detailed below, for some applications it would have been desirable if oligos were synthesized with a free 3xe2x80x2-end, but so far, stepwise synthesis in a 5xe2x80x2xe2x86x923xe2x80x2 direction is problematic. By applying methods developed for the construction of microprocessors, in situ synthesis of arrays of very high complexity has been achieved. The devices can be manufactured at limited cost and are already in use experimentally to investigate nucleic acid samples in order to distinguish and quantitate target sequences. Because the reagents are constructed in situ, it is not possible to ensure that individual oligonucleotides are of full length and without defects. With step-wise synthesis yields of considerably less than 100%, further compounded by the risk that oligonucleotides are damaged during synthesis by light or low pH, arrayed oligonucleotides are contaminated with truncated variants, significantly affecting analyses.
Specificity of Array-Based Analyses
Most commonly, oligonucleotides immobilized in arrays are employed to interrogate a nucleic acid sample on the basis of the differential hybridization stability of target molecules that are perfectly base-paired to an immobilized probe, versus ones that are mismatched in one or more nucleotide positions. This analysis can be enhanced by using very large sets of probe oligonucleotides that include many or most of the sequence variants that can be foreseen in a target sequence Moreover, the target sequence to be analysed can be mixed with equimolar amounts of a differentially labelled target sequence of known composition, to serve as an internal control in the analysis.
Besides DNA base-pairing, several molecular genetic assays also enlist the help of nucleic acid-specific enzymes for increased power of distinction among target sequence variants, or to identity rare target sequences in complex samples. Examples of such assays include ones taking advantage of the reduced efficiency of a primer, mismatched at its 3xe2x80x2 end, to be extended by a DNA polymerase. This technique is used for DNA sequence variant distinction in methods variously referred to as allele-specific amplification, amplification refractory mutation screening, primed amplification of specific alleles, etc.
Polymerases can also be applied to distinguish target sequence variants by determining which nucleotide is incorporated at the 3xe2x80x2 end of a primer, hybridizing just upstream of a variable nucleotide position, known as minisequencing or primer extension. Primer extension reactions have been used to analyze target sequences with single probes and with probes immobilized in arrays, as well as in situ (PRINS).
Both of the two classes of methods where polymerases are combined with hybridization probes require that immobilized probes have a free 3xe2x80x2 end to be extended by the polymerase-assisted incorporation of nucleotides. They serve to efficiently distinguish among closely similar target sequence-variants. The enzyme-assisted extension also offers somewhat increased specificity of target recognition, since it is particularly important that the 3xe2x80x2 end of the primer is correctly base-paired to the target sequence for the extension reaction to take place, adding to the specificity of target recognition in complex samples.
Another class of methods is based on the template-dependent joining of the 5xe2x80x2 and the 3xe2x80x2 end of probe molecules by a ligase. As with polymerases, this strategy places strict requirements on the basepairing of the two juxtaposed oligonucleotide ends to be joined, offering efficient distinction among related target sequence variants. The strategy also provides highly specific recognition of target sequences, even in complex DNA samples, by virtue of the requirement that two probe segments hybridize to the target sequence samples of techniques based on the use of ligases for sequence distinction include the oligonucleotide ligation assay, the ligase chain reaction, and padlock probes.
For reasons listed above, it would be very advantageous to work with arrays of the reversed, 5xe2x80x2xe2x86x923xe2x80x2 oligonucleotide orientation. As mentioned, synthesis of oligonucleotides of this orientation is difficult, and proceeds with substantially lower yield. This problem is even more serious for oligonucleotides to be synthesized in situ.
According to the present invention, the above problem is overcome by a method of preparing immobilized ologonucleotides having free 3xe2x80x2-ends, which method comprises the steps of:
(i) preparing an oligonucleotide attached in a first position to a solid support via its 3xe2x80x2-end and having a free 5xe2x80x2-end;
(ii) binding said oligonucleotide in a second position remote from the 3xe2x80x2-end to the solid support; and
(iii) selectively releasing the 3xe2x80x2-end of the oligonucleotide from the solid support to obtain the oligonucleotide attached to the support in said second position in a reversed orientation with a free 3xe2x80x2-end.
In the following, the method of the invention will be described in more detail.